Based on recent findings which demonstrate the capacity of confluent monolayers of H4-II-E-C3 cells (Reuber cells) to respond to vitamin K in a manner highly reminiscent to that of vitamin K-deficient and anticoagulant-treated rats, studies designed to investigate the biosynthesis and subsequent modification of preprothrombin in cell culture are proposed. The biosynthesis of preprothrombin in cultured cells will be investigated from the standpoint of identifying and characterizing potential regulatory mechanisms associated with the transcription and translation of preprothrombin mRNA as well as those posttranslational modifications required for prothrombin secretion and subsequent enzymatic activation. Antibodies specific for rat prothrombin and preprothrombin will be used to isolate (affinity chromatography) and quantitate (radioimmunoassay) the prothrombins synthesized in Reuber cells in response to vitamin and various serum supplements. Prothrombins isolated by affinity chromatography and/or barium adsorption and obtained from both extracellular- and intracellular-fractions of Reuber cell cultures will be further characterized by SDS-and urea-gel electrophoresis. In conjunction with pulse and pulse-chase labeling systems, these procedures will provide an accurate assessment of prothrombin biosynthesis, modification and degradation. An assessment of preprothrombin mRNA will be made following its isolation by translation in a cell-free wheat germ system and measurement of the amount of labeled prothrombin synthesized. Fluctuations in the metabolism of prothrombin, observed in the presence of a host of anticoagulants, antimetabolites and antibiotics should aid in elucidating the regulatory controls imposed upon prothrombin biosynthesis in cultured liver cells.